Proteases and uses thereof

Patent metadata

Field	Value
Publication number	US-11549105-B2
Application number	US-201816955088-A
Country	US
Kind code	B2
Filing date	Dec 21, 2018
Priority date	Dec 21, 2017
Publication date	Jan 10, 2023
Grant date	Jan 10, 2023

Abstract

The present invention relates to novel proteases, more particularly to protease variants having improved activity compared to the protease of SEQ ID NO: 1 and the uses thereof for degrading polyester containing material, such as plastic products. The proteases of the invention are particularly suited to degrade polylactic acid, and material containing polylactic acid.

First claim

The invention claimed is:

1. A protease which (i) has at least 90% sequence identity to the polypeptide of SEQ ID NO: 1, and (ii) has at least one amino acid substitution selected from S101F/L/M/W/Y, S103L, T106I/L, G131I, and G133K, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1, and (iii) exhibits increased polyester degrading activity compared to the polyester degrading activity of the protease of SEQ ID NO: 1.

2. The protease according to claim 1 , wherein said protease comprises at least a combination of substitutions selected from S101F/L/M/W/Y+S103L+T106I/L, S101F+S103L, S101F+T106I, T106I+G133K, S101F+S103L+T106I, S101F+S103L+G133K, S101F+T106I+G133K, S101F+S103L+T106L, S101F+S103L+T106I+G133K and S101F+S103L+T106I+G131I.

3. The protease according to claim 1 , wherein said protease further comprises at least one amino acid substitution at a position selected from D12, L21, T175, S194, H197, G212, I217 and R247 wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

4. The protease according to claim 1 , wherein the protease further comprises at least one amino acid substitution or combination of substitutions selected from D12C+L21C, T175C+R247C, S194P, H197D, G212N, and I217K, wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO: 1.

5. The protease according to claim 1 , wherein said protease exhibits an increased polyester degrading activity compared to the protease of SEQ ID NO: 1 at pH between 7 and 10.

6. The protease according to claim 1 , wherein said protease exhibits an increased polyester degrading activity compared to the protease of SEQ ID NO: 1 at pH 7.5 and pH 9.

7. The protease according to claim 1 , wherein said protease has at least 95% sequence identity to the polypeptide of SEQ ID NO: 1.

8. The protease according to claim 1 , wherein said protease has at least one amino acid substitution selected from S101F/L/M/W/Y, S103L and T106I/L.

9. A composition comprising the protease of claim 1 .

10. A plastic compound comprising (i) at least one polyester and (ii) the protease of claim 1 .

11. The plastic compound according to claim 10 , wherein the protease is able to degrade at least one polyester of the plastic compound.

12. The plastic compound according to claim 10 , wherein the polyester comprises polylactic acid.

13. A process for producing a plastic compound comprising mixing a polyester and the protease of claim 1 at a temperature at which the polyester is in a partially or totally molten state.

14. The process according to claim 13 , wherein the polyester and the protease are extruded to form a plastic compound.

15. A method of degrading a plastic product containing at least one polyester comprising:
(a) contacting the plastic product with the protease according to claim 1 , and
(b) recovering monomers and/or oligomers.

16. The method according to claim 15 , wherein the plastic product comprises at least one polyester selected from polylactic acid (PLA), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polyethylene terephthalate (PET) polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA) and blends/mixtures of these materials.

Assignees

• Carbios

Inventors

• Marty Alain
• Duquesne Sophie
• Guicherd Marie
• Gueroult Marc
• Andre Isabelle

Classifications

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C12N9/52 Primary

  derived from bacteria {or Archaea} · CPC title

• Y02W30/62

  Plastics recycling; Rubber recycling · CPC title